POMC调控乳腺癌脑转移的作用及其机制研究

Cancer stem cells are thought to be closely related to brain metastases. The applicant has established two breast cancer stem cell (BCSC) lines that can be cultured in vitro for long periods from breast cancer tissues (Patent Application No. 201510330647.5), confirming that POMC (pro-opiomelanocortin) is a key molecule that maintains BCSC properties (Int J Cancer 2017). It has been reported that SOX2 is associated with BCSC. Through gene chips and validation analysis, the applicant found that POMC and SOX2 were highly expressed in brain metastases of breast cancer, and their expression was positively correlated. Therefore, the applicant hypothesized that POMC may enhance breast cancer stemness and brain metastasis by up-regulating SOX2 expression. This project intends to use CRISPR / Cas9, in vitro blood-brain barrier model and in vivo animal imaging technology to confirm that POMC enhances the stemness of BCSC and promotes BCSC colonization and growth in brain tissue by up-regulating SOX2 expression, and to explore the molecular mechanism and clinical relevance of POMC in regulating brain metastases of breast cancer. The implementation of this project will initially clarify the role of POMC in enhancing breast cancer stemness and brain metastasis and provide theoretical basis and clinical application basis for the occurrence, development, prognosis and accurate targeted therapy of breast cancer brain metastasis.

肿瘤干细胞被认为与脑转移关系密切。申请者从乳腺癌组织中建立了可长期体外培养的乳腺癌干细胞（BCSC）系（专利申请号201510330647.5），证实POMC（阿片促黑素细胞皮质素原）是维持BCSC特性的关键分子（Int J Cancer 2017）。已报道，SOX2与BCSC相关。申请者通过基因芯片及验证分析发现POMC和SOX2在乳腺癌脑转移组织中高表达并呈正相关。因此，申请者提出POMC可能通过上调SOX2表达，增强乳腺癌干性和脑转移。本项目拟采用CRISPR/Cas9、体外血脑屏障模型和活体动物成像等技术，明确POMC通过上调SOX2表达，增强BCSC的干性，促进BCSC在脑组织中定植和生长；探讨POMC调控乳腺癌脑转移的分子机制及临床相关性。本项目的实施将初步阐明POMC增强乳腺癌干性和脑转移的作用机制，为乳腺癌脑转移的发生发展、预后判断及精准靶向治疗提供理论基础和临床应用依据。

乳腺癌是全世界范围之内女性最高发的恶性肿瘤，常见的远处转移部位包括骨、肺、肝和脑，其中脑转移的发生率大约为15%-20%。 HER2阳性和三阴性乳腺癌是易发生脑转移的高危亚型，严重威胁患者的生命，是乳腺癌死亡的重要原因。我们取3对乳腺癌脑转移组织和乳腺癌组织，采用基因芯片结果提示89个基因的表达在脑转移瘤中表达上调，而230个基因在脑转移瘤中表达下调。生物信息学分析发现POMC、SOX2、CORO2A等基因在本中心的临床脑转移标本和GEO数据库实验性动物脑转移中上调。扩大样本验证，SOX2和CORO2A在脑转移灶中和乳腺癌组织中差异显著，但现POMC和SOX2在乳腺癌脑转移组织中高表达并呈正相关。SOX2的表达高低与无乳腺癌脑转移生存密切相关。用SOX2过表达与干扰慢病毒建立了稳定表达SOX2的231-SOX2细胞及对照231-Control细胞，建立了SOX2敲低的1806-shSOX2及对照细胞。过表达SOX2促进乳腺癌细胞的增殖和侵袭，干扰SOX2的表达则抑制侵袭和转移。SOX2过表达促进乳腺癌细胞粘附HBMEC，跨HBMEC内皮迁移和跨体外血脑屏障迁移，而敲低SOX2则观察到相反的效果。 SOX2的表达与脑转移特征相关基因HBEGF和FSCN1的表达呈正相关。在mRNA和蛋白水平，SOX2过表达上调HBEGF和FSCN1的表达，敲低SOX2则下调HBEGF和FSCN1的表达。此外，高水平的FSCN1和HBEGF也与乳腺癌患者的无脑转移生存率显着相关。机制上，SOX2可激活AKT和β-catenin信号通路分别调控HBEGF和FSCN1的表达，SOX2敲低和AKT抑制在下调HBEGF的表达方面可达到类似的效果。SOX2敲低和β-catenin抑制也可以达到类似的抑制FSCN1表达的效果。左心室注射成瘤实验发现过表达SOX2促进乳腺癌脑转移的发生，敲低SOX2则观察到相反的效果。我们发现多个基因在乳腺癌脑转移瘤中差异表达，经过鉴定SOX2的统计学差异最为显著。SOX2的表达升高意味着较短的无脑转移生存时间。SOX2可能通过激活AKT和β-catenin信号通路分别调节HBEGF和FSCN1的表达来促进乳腺癌脑转移。本项目阐明POMC增强乳腺癌干性和脑转移的作用机制，为乳腺癌脑转移的发生、预后判断及精准靶向治疗提供理论基础和临床应用依据。